Most eukaryotes possess a cellular defense system for protecting their genomes against invading foreign genetic elements. Insertion of foreign elements is believed to be generally accompanied by formation of double-stranded RNA (dsRNA) that is interpreted by the cell as a signal for unwanted gene activity. dsRNA induces potent and specific gene silencing through a mechanism referred to as RNA interference (RNAi) in animals or posttranscriptional gene silencing (PTGS) in plants. The currently accepted mechanism commences with processing of the long dsRNA by Dicer RNase III into small interfering RNAs (siRNAs) that are double-stranded and are predominantly 21 or 22 nucleotides in length. Thereafter, siRNA duplexes are assembled into a multi-component complex, called the RISC complex (RNA-induced silencing complex), which guides the sequence-specific recognition, hybridization, and cleavage of endogenous mRNA.
One of the components of RISC is EIF2C2, a member of the large family of Argonaute proteins that are characterized by the presence of a PAZ domain and a C-terminal Piwi domain (Hammond et al., Science, 2001, 293, 1146-1150). Although these domains have an unknown function, it has been suggested that the PAZ domain may act as a protein-protein interaction motif. Dicer also contains a PAZ domain and this may provide a mechanism for interaction between Dicer and EIF2C2 to facilitate siRNA incorporation into RISC (Martinez et al., Cell, 2002, 110, 563-574). Other members of the Argonaute family have been implicated in translational control, microRNA processing, and development in diverse species (Carmell et al., Genes Dev., 2002, 16, 2733-2742).
The gene encoding human EIF2C2 was first cloned in 1999 during the course of cloning EIF2C1 (Koesters et al., Genomics, 1999, 61, 210-218). EIF2C1 is a eukaryotic protein translation initiation factor that forms part of the GTP-binding complex and assists with methionyl-tRNA(i) binding to the 40S ribosomal subunit. EIF2C2 (also called Ago2, Argonaute 2, and eukaryotic translation initiation factor 2C,2) was accidentally isolated as a crosshybridizing cDNA clone and found to be 85% identical to EIF2C1 which is frequently lost in human cancers such as Wilms tumors, neuroblastoma, and carcinomas of the breast, liver, and colon. The gene encoding EIF2C2 was again cloned in 2001 when the protein was sequenced and it was identified as a member of the Argonaute family and as a homolog of rde-1, a protein essential for RNAi in C. elegans (Hammond et al., Science, 2001, 293, 1146-1150).
EIF2C2 has been identified as a component of RISC and may facilitate siRNA incorporation into RISC, however EIF2C2 may also have other biochemical functions. For example, a common form of inherited mental retardation called fragile X syndrome is caused by the loss of FMR1 expression, a gene which encodes an RNA-binding protein. The FMR1 complex in Drosophila contains two ribosomal proteins, 5S RNA, and EIF2C2. Since the Drosophila FMR1 complex can also associate with Dicer, a model has been suggested for translation control in Drosophila whereby the RNAi and FMR1 pathways intersect and raise the possibility that defects in the RNAi-related machinery may cause human disease (Caudy et al., Genes Dev., 2002, 16, 2491-2496; Ishizuka et al., Genes Dev., 2002, 16, 2497-2508).
EIF2C2 has also been found in a large 15 subunit ribonucleoprotein (RNP) that contains Gemin3, Gemin4, and numerous microRNAs. RNPs are assembled and restructured by the Survival of Motor Neurons (SMN) complex, a complex which results in the neurodegenerative disease spinal muscular atrophy when its levels are reduced (Mourelatos et al., Genes Dev., 2002, 16, 720-728).
EIF2C2 and Dicer have also been found to be mislocalized to perinuclear regions of germline cells in mutant ovaries of Drosophila with a null allele of maelstrom. As maelstrom is a component of Drosophila nuage, the perinuclear granules that are a hallmark of germline cells across the animal kingdom, the mislocalization of EIF2C2 and Dicer in maelstrom mutants suggests a potential connection between nuage and the microRNA or RNAi pathways (Findley et al., Development, 2003, 130, 859-871).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of EIF2C2. Suppression of EIF2C2 expression in Drosophila S2 cells with two different double-stranded RNA approximately 1000 nucleotides in length was used to demonstrate that EIF2C2 is essential for RNAi in these cells (Hammond et al., Science, 2001, 293, 1146-1150).
Consequently, there remains a long felt need for agents capable of effectively inhibiting EIF2C2 function. These agents would serve to mediate RNAi pathways and could potentially serve to ameliorate disease conditions associated with aberrant RNA processing or metabolism.
The present invention provides compositions and methods for modulating EIF2C2 expression and consequently the RNAi pathway.